PROJECT SUMMARY Retinopathy of prematurity (ROP) is a vasoproliferative retinopathy and a leading cause of visual impairment and blindness in children worldwide. Vision loss from ROP occurs as a result of oxygen-induced damage of retinal vessels and a delay in physiologic retinal vascular development (PRVD) in Phase I, which leads to pathologic vessel growth out of the retina and into the vitreous, intravitreal neovascularization (IVNV) in Phase II. Treatment of ROP includes laser ablation of the hypoxic avascular retina and, more recently, angiogenic inhibitors against vascular endothelial growth factor (VEGF) to reduce IVNV. Clinical studies report that extremely premature infants?even infants who never developed ROP? do not develop normal vision and that 33% of infants with ROP developed glaucoma as children, a disease that causes degeneration of retinal ganglion cells (RGCs). The use of anti-VEGF agents in humans with diabetic retinopathy thins retinal layers containing RGC axons and increases the need for glaucoma surgery. These observations associate premature birth, VEGF inhibition and RGC injury, which all contribute to vision loss. And, the objective of this research is to understand the mechanisms involved. Besides its role in angiogenesis, VEGF has been implicated as a factor that protects RGCs in ischemia, stimulates RGC axon growth in retinal explant cultures and guides RGC axon development in the brain during development. VEGF is increased in ROP and in ROP animal models. Using a rat oxygen-induced retinopathy (OIR) model representative of ROP, our previous work found that Mller cell (MC)-specific lentiviral knockdown of upregulated VEGF reduced IVNV without interfering with PRVD, but also thinned the neural retina, increased neural apoptosis, and increased neuroprotective factors associated with RGC damage. In contrast, endothelial cell (EC)-specific lentiviral knockdown of VEGF receptor 2 (VEGFR2) reduced IVNV, increased PRVD and increased the thickness of the neural retina. Our preliminary data found OIR reduced RGC numbers and dendrites projections and that reducing upregulated VEGF further reduced RGC numbers whereas regulating VEGFR2 in endothelial cells increased RGC numbers. Based upon clinical observations and these data, we propose the hypothesis that developing RGCs exposed to oxygen stresses of prematurity require upregulated VEGF as a neuroprotective factor and to promote proper integration of RGCs into retinal circuits in order to improve visual function. Aim 1 will be to clarify the roles of upregulated VEGF in developing RGCs and for visual function under oxygen stresses of prematurity. Aim 2 will be to determine if regulating VEGFR2 signaling in ECs is sufficient to allow development of RGCs and visual function under oxygen stresses of prematurity. Aim 3 will be to identify VEGF signaling mechanisms in RGCs that promote neuroprotection and neurite growth under oxygen stresses of prematurity. Methods include: rat OIR model of ROP; lentiviral gene therapy; immunohistochemistry; optical coherence tomography imaging; optomotor reflex-based behavioral tests to measure visual outcomes; and primary RGC culture.